Since the early 1950's, it has been known that the presence of bacterial organisms in the gastro-intestinal tract is accompanied by a high concentration of urease, which hydrolyses urea to form carbon dioxide and ammonia. These gases are detected in the subject's blood stream and ultimately, in the subject's breath, if he had been administered isotopically labeled urea. Such early results appear in reviews published by R. W. VonKorff et al. in Am. J. Physiol., Vol. 165, pp. 688-694, 1951, and by H. L. Kornberg and R. E. Davies in Physiol. Rev., Vol. 35, pp. 169-177, 1955.
Since these early experiments, it has been found that there exist, in addition to the bacterial infections initially studied, a significant number of medical conditions associated with disorders of the gastro-intestinal tract or metabolic or organ malfunctions, which are capable of detection by means of such simple breath tests. These breath tests are based on the ingestion of an isotopically labeled sample, which is cleaved by the specific bacteria or enzyme action being sought, or as a result of the metabolic function being tested, to produce labeled gaseous by-products. These by-products are absorbed in the blood stream, and are exhaled in the patient's breath, where they are detected by means of external instrumentation.
Though the early experiments were performed using the radioactive carbon-14 atom, the most commonly used atom in such test procedures today is the carbon-13 atom, which is a stable, non-radioactive isotope, present in a proportion of about 1.1% of naturally occurring carbon. The labeled substance contains the functional compound to be used in the test, with almost all of its 12C atoms replaced by 13C atoms. Enrichments of up to 99% of 13C are typically used. This compound is cleaved enzymatically under the specific conditions being tested for, either during gastric absorption, or during gastro-intestinal transit, or during its metabolisation in other organs of the body. The cleavage product produced is 13CO2, which is absorbed in the bloodstream and exhaled in the patient's breath together with the CO2 naturally present. The breath sample is then analyzed, usually in a mass spectrometer or a non-dispersive infra-red spectrometer. The increased presence of 13CO2 is determined, as compared with the expected 1.1% of total CO2 in healthy patient's breath, resulting from the metabolism of carbon compounds with the naturally occurring level of approximately 1.1% of carbon-13.
Though carbon-13 is the most commonly used isotropic replacement atom in such breath tests, other atoms which have been used include nitrogen-15 and oxygen-18. In addition, carbon-14 is still used in some procedures, but being radioactive, there are severe disadvantages both to its ingestion by the patient, and because of the storage, handling and disposal precautions required at the test site.
There are an increasing number of metabolic disorders, bacterial infections and organ malfunctions which can be diagnosed using such labeled substances for enabling breath tests. New applications are being proposed continuously, but among the more common currently in use are:                (a) The detection of Helicobacter pylori infections gastric and duodenal tracts, by means of the ingestion of 13C-labeled urea and breath detection of an increased level of 13CO2. It is also feasible to use 15N-labeled urea, and to detect nitrogen-15 ammonia 15NH3 in the breath, but this test format is not currently in use. Gastric and duodenal ulcers, non-ulcer dyspepsia and gastritis have been shown to be related to the presence of Helicobacter pylori infections.        (b) The detection of fat malabsorption, such as is present in steatorrhea and Crohn's disease, by means of the ingestion of 13C-labeled triolein or tripalmitin, and breath detection of an increased level of 13CO2.        (c) Liver function evaluation (by monitoring the P450 enzyme activity), liver disease severity and detoxification activity by means of the ingestion of 13C-labeled aminopyrin, methacitin or caffeine citrate (depending on the specific function being tested) and breath detection of an increased level of 13CO2.        (d) The measurement of hepatic mitochondrial activity by means of the ingestion of 13C-labeled octanoic acid, and breath detection of an increased level of 13CO2.        (e) A check of hepatic mitochondrial function efficiency by means of the ingestion of 13C-labeled ketoisocaproic acid, and breath detection of an increased level of 13CO2.        (f) The quantification of functional liver mass by means of the ingestion of 13C-labeled galactose, and breath detection of an increased level of 13CO2.        (g) The testing of gastric emptying function by means of the ingestion of 13C-labeled octanoic acid for the emptying rate of solids, or 13C-labeled sodium acetate for the emptying rate of liquids, and breath detection of an increased level of 13CO2.        (h) The determination of exocrine pancreatic insufficiency by means of the ingestion of a 13C-labeled mixed triglyceride sample such as octanoil-1,3-distearin for checking the lipase function, or a 13C-labeled sample of corn starch for checking the amylase function, and breath detection of an increased level of 13CO2. The mixed triglyceride test is one of the tests used for detecting cystic fibrosis. For the evaluation of the digestion and absorption of medium-chain fatty acid triglycerides, 13C-labeled trioctanoin is used in preference to the mixed triglyceride.        (i) The detection of bacterial overgrowth in the small intestine by means of the ingestion of 13C-labeled glycolic acid or xylose, and breath detection of an increased level of 13CO2.        (j) The testing of lactose or glucose intolerance, by means of the ingestion of 13C-labeled lactose or glucose, and measurement of the speed of appearance of an increased level of 13CO2 in the breath.        
Previously available tests for these illnesses generally involve drastically more invasive procedures, and are therefore much less patient compliant than the simple breath tests described above. Such procedures include gastro-endoscopy, with or without the removal of a tissue biopsy, biopsies or organs suspected of malfunction, blood tests to detect antibodies to suspected bacteria, blood biochemistry tests following ingestion of suitable compounds, and radiological tests, whether by gamma imaging of the organ function following ingestion or injection of a suitable gamma emitter, or by direct X-ray imaging or CT scanning. Furthermore, there are other disadvantages to the previously used tests, such as the fact that they rarely give real time information about the organ function or status being observed. In some cases, such as in the case of blood tests for antibodies of bacterial infections, they give historic results which may have no therapeutic relevance currently, since antibodies to a particular bacterium can remain in the body for up to 2 years from the date that the infection has been eradicated.
The above mentioned breath tests are completely non-invasive, and are executed in comparative real time, so that they have a great advantage over previously available tests, and their use is gaining popularity in the medical community, as evidenced by the fact that suitable isotopically labeled substances are currently available commercially from a number of sources.
However, in spite of the advantages of isotopically labeled breath tests, current instrumentation and procedures for performing it sill have a number of serious drawbacks, which continue to limit its usefulness. The major disadvantage, which becomes apparent when a review of prior art breath test performance techniques and instrumentation is performed, is that none of the currently used techniques are sufficiently rapid to permit immediate measurement of the requested parameter, allowing a diagnosis for the patient in a single short visit to the physicians office.
One of the early breath tests to be proposed is that for detecting the presence of the Helicobacter pylori bacterium in the upper gastro-intestinal tract, by means of the oral administration of isotopically labeled urea, and the detection of the presence of isotopically labeled carbon dioxide or ammonia in the patient's breath resulting from the hydrolysis of the urea by the urease which always accompanies H. pyroli infections. This method is described by Marshall in U.S. Pat. No. 4,830,010. In this implementation of the test, the breath of the subject is collected, preferably from 10 to 120 minutes after administration of the substance, in a balloon inflated by the subject, and from there is transferred to a storage and transport container, such as a Vacutainer® sold by Becton-Dickenson Inc.
According to a method proposed by Marshall, the sample is then analysed by mass spectrometry or by infra-red or nuclear magnetic resonance spectroscopy, for the presence of isotopically labelled CO2 resulting from the hydrolysis of the urea. If the radioactive carbon-4 is used to label the urea, then the breath sample is analysed by bubbling it through a scintillation solution, which is transferred to a scintillation counter to determine the presence of beta radiation in the exhaled breath specimen. Because of the cost and complexity of the analysis instrumentation, in none of the preferred methods described by Marshall is it suggested that the analysis of the breath may be performed on site at the point where the sample is taken from the patient. The subject must thus wait at least ten minutes to give the sample, and must then wait for the laboratory to return the results. Clearly this method cannot be used to provide the results of the test within the context of a single visit to the office of the physician.
In a recent article entitled “Minimum Analysis Requirements for the Detection of Helicobacter pylori Infection by the 13C-Urea Breath Test” by P. D. Klieg and D. Y. Graham, published in Am. J. Gastroenterol., Vol. 88, pp. 1865-1869, 1993, a statistical study of the reliability and minimum criteria for conducting this test is presented. The breath analyses were again performed by gas isotope ratio mass spectrometry at a remote site. Amongst their findings are that breath sampling at 30 minutes after urea ingestion is likely to lead to significantly less false-positive and false-negative results, than sampling after 20 minutes, and that sampling after 30 minutes is therefore their proposed protocol time. They also conclude that “In the current environments of clinical research and patient care, the costs and turnaround times of CO2 isotropic abundance measurements continue as the major barriers to commercial propagation of the 13C-urea breath test.”
In another described prior art method of executing the urea breath test, Koletzko and co-workers describe the analysis of the exhaled breath by means of an isotope-selective non-dispersive infrared spectrometer [Koletzko et al., Lancet, 345:961-2, 1995]. Even using such a sophisticated instrument, the subjects are still required to wait 15 and 30 minutes for successive breath samples to be taken. Such a long delay to obtain breath samples, as well as the long wait between samples, is inconvenient and potentially reduces patient compliance.
Furthermore, as in the previously mentioned prior art, the sample or samples are collected from the patient and then sent to a laboratory for analysis, causing a delay in the determination of the results and forcing the subject to return to the office of the physician to obtain the results. If the test does not yield meaningful results, the entire process must be repeated again. The requirement for multiple office visits potentially further reduces patient compliance. The potential reduction in patient compliance can have serious consequences, since Helicobacter pylori is implicated by the World Health Organisation as a possible cause of stomach cancer, in addition to its role in gastric and duodenal ulcers.
The most rapid breath test currently proposed, the “Pytest” from Tri-Med Specialities, Charlottesville, N.C., USA, takes about 10-15 minutes to perform but uses radioactive carbon-14 isotopically-labeled urea [D. A. Peura, et al., Am. J. Gastro., 91:233-238, 1996]. The presence of 14CO2 in the subject's exhaled breath is detected by direct beta counting. This test thus has all the disadvantages of the use of radioactive materials. Not only is the ingestion of radioactive materials potentially hazardous, but it also restricts the test to large testing centers which can handle such materials. Thus, the test cannot be performed in the office of the average physician, so that multiple office visits are again required.
Another recent prior art method which discusses implementations of the 13C-urea breath test, is shown in PCT Application No. WO97/14029, entitled “Method for Spectrometrically Measuring Isotopic Gas and Apparatus thereof”, applied for by the Otsuka Pharmaceutical Company of Tokyo, Japan. In this application too, the exhaled breath sample is transferred in sample bags from the patient to the spectrometer, which, because of its cost, complexity and size, has perforce to be installed in a central sample collection laboratory, and not in the doctor's office or near the patient's bed. The inventors in fact state that “The measurement of such breath samples is typically performed in a professional manner in a measurement organisation, which manipulates a large amount of samples in a short time.” This prior art proposes the use of one breath sample before the administration of the urea, and another after a lapse of 10 to 15 minutes.
Other prior art which describe sensitive analyzer systems for measuring the isotopic ratios of 13CO2 to 12CO2 in a gaseous sample, such as is required in an exhaled breath analyzer for performing the above mentioned breath tests, includes U.S. Pat. No. 5,077,469, granted to W. Fabinski and g. Bernhardt, which describes a double reference path non-dispersive infra-red gas analyzer. A further development of such an instrument described in European Patent Application No. EP 0 584 897 A1 can be used to compare the two isotopic CO2 concentrations in the exhaled breath by means of infra-red absorption measurements on two IR-cells filled with gas from the same breath sample.
In U.S. Pat. Nos. 4,684,805 and RE 33493, granted to P. S. Lee, R. F. Majkowski and D. L. Partin, an infra-red absorption spectrometer is described for discriminating between the two isotopic CO2 molecules for the breath tests. Their spectrometer design uses lead salt laser diodes as the source of radiation. Such laser diodes have emission lines in the 4 μm to 5 μm wavelength region of the infra-red spectrum, where the strongest CO2 absorption lines are located. As a consequence, despite the lack of temperature stability of such laser diodes, and the fact that they must be operated at liquid nitrogen temperatures, their use enables the spectrometer to achieve the high selectivity and sensitivity required for breath test analysis.
U.S. Pat. No. 5,317,156, granted to D. E. Cooper, C. B. Carlisle and H. Riris, describes an FMS (Frequency Modulation Spectroscopy) laser absorption spectrometer for distinguishing between the week 12CO2 and 13CO2 absorption lines in the 1.6 μm infra-red region, where highly stable laser diodes are available. Even though the CO2 lines are very weak in this region, the stability of the GaAs laser diodes used as the source in this range, and the sophisticated TTFMS (two-tone Frequency Modulation Spectroscopy) technique used enables the inventors to provide sufficient differentiation between the two isotopes of CO2 that the spectrometer can be used in breath test analysis.
In U.S. Pat. No. 5,394,236, granted to D. E. Murnick, an apparatus for isotopic analysis of CO2 is described by means of laser excited spectroscopy, utilising the optogalvanic effect to differentiate between the light of different wavelengths.
Because of the need to provide high sensitivity and good mass discrimination, all of the above described analysis systems are complex in nature. They are therefore, costly to manufacture and generally of large dimensions, making them suitable for commercial exploitation only for large and high sample volume installations.
A number of commercial companies offer complete systems for performing breath tests for the detection and study of the various gastro-enterologic conditions mentioned previously, using the isotopically labeled substances commercially available.
The Alimenterics Company of Morris Plains, N.J., markets the Pylori-Chek 13C-Urea breath test kit for use with its LARA™ System, for detecting the presence of H. Pylori in the gastro-intestinal tract. The company is developing kits for the clinical use of the other breath tests mentioned above. Breath is collected in a uniquely designed breath collection device, that also serves to transport the sample to the LARA™ System. This system, which stands for Laser Assisted Ratio Analyzer, is a sophisticated infra-red spectrometer designed to provide the sensitivity required to detect tiny percentage changes in the level of 13CO2 in the patient's exhaled breath. Because of the complexity of the LARA™ System, it is a large piece of equipment, weighing over 300 kg, and very costly. Consequently, this system too is only feasible for very large institutions and central laboratories, where the large number of tests performed can justify the cost.
Meretek Diagnostics Incorporated of Nashville, Tenn., has also developed such a 13C-Urea breath test diagnostic system, and use an isotopic ratio mass spectrometer called the ABCA (Automated Breath 13C Analyzer) manufactured by Europa Scientific Limited, of Crewe, Cheshire, U.K. for analyzing the breath samples. In this system too, the analyzer unit is large, costly and sophisticated, and therefore is usually located remote from the collection point.
Wagner Analysen Technik GmbH of Worpswede, Germany, offers an infra-red non-dispersive spectrophotometer-based system called the IRIS®—Infra Red Isotope Analyser, which is based on the above-mentioned European Patent Application No. EP 0 584 897 A1. Though the main useage mode is by means of transport of the breath samples from the collection point to the analyzer in sample bags, this system, according to the manufacturer's sales literature, also has a sample port whereby connection can be made directly to a breathing mask, an incubator, or a breathing machine. No details of such a connection tube accessory are however given in the technical manual accompanying the analyzer, nor does the manufacturer provide any programs with the system's operational software to enable such an accessory to be used for performing on-line analyses. This analyzer has dimensions of 510×500×280 mm and weighs 12 kg., and in addition, a PC is required for control. Though smaller and less costly than those mentioned above, it is still too large and heavy to be described as a truly portable device. Furthermore, its reported cost of several tens of thousands of U.S. Dollars, though considerably less than that of the two above-mentioned commercial systems, still makes it unsuitable for point-of-care or physician's office use.
In the preferred procedures described in all of the above mentioned prior art, the patient must wait typically 20-30 minutes before the active sample is collected, mainly because only one sample is taken beyond a background sample. This time is necessary to allow the level of isotopically labeled exhaled gas to reach a relatively high value, close to its end value, to enable the analyzer to measure the gas with a sufficient confidence level. However, such a single point determination potentially decreases the accuracy of the test, as well as increasing the risk of ambiguous results.
To the best of our knowledge, no breath test analyzer system has been described in the prior art which is sufficiently small, fast in producing reliable results, low in production cost, portable and sensitive, to enable it to be used as for executing tests in real time in the physician's office or at another point of care.